Since a few years there is a great interest in the biotechnological field to modify naturally occurring antibodies in order to obtain more specified and more individual antibody species. Therefore, attempts have been made to produce (modified) antibody fragments.
All naturally occurring antibodies of all classes have at least two binding sites. This enables them to bind to a surface with a greater functional affinity (also called avidity) than monovalent fragments, such as Fab fragments. Over the last few years, methods have been described (Skerra and Pluckthun, 1988, Science 240, 1038-1040; Better et al., 1988, Science 240, 1041-1043) with which functional antibody fragments can be produced in Escherichia coli. These include the Fv fragment (the heterodimer consisting of V.sub.H and V.sub.L) and the Fab fragment (consisting of the complete light chain with the domains V.sub.L and C.sub.L as well as the first two domains of the heavy chains V.sub.H and C.sub.H1).
The Fv fragment, however, has a tendency to dissociate into V.sub.H and V.sub.L and therefore, it is advantageous to link the two domains covalently. One particular way of linking them is by designing a peptide linker between them, either in the orientation V.sub.H -linker-V.sub.L or V.sub.L -linker-V.sub.H (Bird et al.,1988, Science 242, 423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879) The resulting fragments are called single-chain Fv fragments.
All these fragments are, however, monovalent. We describe in this invention a method to engineer small dimerization domains based on peptides forming amphipathic helices. These peptides will be referred to as "intercalating", but this term is only meant to express the ability of targeted association and not a restriction referring to a particular structure of the dimerization interface.
While the methodology described here, is in principle applicable to either Fab, Fv or scFv fragments, it is the latter for which their use is most advantageous. In this case bivalent fragments can be constructed of very small size, and still the dissociation into V.sub.L and V.sub.H as well as the wrong matching of fragment chains, e.g. V.sub.L --V.sub.L, can be prevented.
Antibody fragments of small size are of particular advantage in many applications. In diagnostic applications (e.g. ELISA, RIA, etc.), the smaller molecules surface decreases the problems of nonspecific interactions, which are known to frequently involve the constant domains. The same is true in using antibody fragments as ligands in affinity chromatography. In tumor diagnostics or therapy, it is important that a significant proportion of the injected antibody penetrates tissues and localizes to the tumor, and is dependent on the molecular dimensions (Colcher et al., 1990, J. Natl. Cancer Inst. 82, 1191-1197). Expression yields and secretion efficiency of recombinant proteins are also a function of chain size (Skerra & Pluckthun, 1991, Protein Eng. 4, 971) and smaller proteins are preferred for this reason. Therefore, molecules of a small size are advantageous for several reasons.
Previously, decreasing the molecular dimensions of the antibody meant the preparation of proteolytic fragments. The smallest bivalent fragments, (Fab)'.sub.2 fragments, are still about twice the size of the present fragments of this invention. Therefore, these new fragments combine three features: (a) small size, (b) bivalence or bifunctionality and (c) ability of functional expression in E. coli.
There is great interest in bifunctional antibodies in a large number of areas. Bifunctional antibodies may be defined as having two different specificities for either two different antigens or for two epitopes of the same antigen.
There are currently a number of methods how to produce bifunctional antibodies. However, none of the existing methods allows to produce exclusively bifunctional antibodies in vivo, but rather a mixture of molecular species always occur, requiring complicated and expensive separation procedures.
Four principal methods can be distinguished. In the first, chemical crosslinking is used, which may advantageously use heterobifunctional crosslinkers. By this method, whole antibodies (Staerz et al., 1985, Nature 314, 628; Perez et al, 1985, Nature 316, 354-356), Fab fragments (Carter et al., 1992, Biotechnology 10, 163) and scFv fragments (Cumber et al., 1992, J. Immunol. 149, 120) have been chemically crosslinked after purification.
The second previous method involved the fusion of two hybridomas to give a so-called heterohybridoma or "quadroma". In this method, any light chain can pair with any heavy chain, and the two heavy chains can give homodimers or heterodimers resulting in very complicated product mixtures (Milstein & Cuello, 1983, Nature 305, 537).
The third method is related to the second and consists of transfecting two expression plasmids into a hybridoma cell, encoding the heavy and light chain of the second antibody (Lenz & Weidle, 1990, Gene 87, 213) or a retroviral vector (De Monte et al., 1990, Acad. Sci. 87, 2941-2945). However, once introduced, the product mixture is identical as in the second procedure.
Finally, antibodies have been reduced, mixed and reoxidized (Staerz & Bevan, 1986, Immunology Today 7). Again, very complicated product mixtures are obtained requiring sophistical separation and quality control procedures.
Thus a method is still needed allowing the isolation of exclusively heterodimeric antibodies directly without the complicated preparation required from chemical crosslinking. In the present invention, this problem is solved by (i) covalently linking corresponding VH and VL domain in a scFv fragment and (ii) using dimerization domains only allowing the formation of heterodimers, such as certain leucine zippers and derivatives.
Another important consideration in the present invention was the desire to make the MW of the bispecific antibody as small as possible for reasons explained above in detail. This was achieved by using scFv fragments.
A number of uses of bispecific antibodies bave been described, and most of them would benefit from this new technology. For example, bispecific antibodies are of great interest in tumor therapy. One arm of the antibody may bind to a tumor marker, the other arm to a T-cell epitope, a toxin, or a radionuclide binding peptide or protein to bring a killing function close to the tumor cell. In diagnostics, one arm may bind to the analyte of interest and the other to a principle which can easily be quantified, e.g. an enzyme. Finally, in cellular applications, it may be advantageous to obtain higher selectivity in binding, if two different epitopes or the same protein complex can be recognized or if two different proteins can be recognized on the same cell surface.
Thus, it was object of the invention to create new individual and stable antibody fragment fusion proteins with bi- or even mulitfunctional binding sites.
It has been found that antibody fragment fusion proteins containing Fv-fragments could be produced by genetic engineering methods which show specified and improved properties.
Object of the invention is, therefore, a monomeric antibody-fragment fusion protein essentially consisting of a Fv-fragment of an antibody and a peptide which is capable to dimerize with another peptide by noncovalent interaction.
The term "noncovalent interaction" means every existing under normal condititions stable linkage which is not related to a covalent binding, for example linkage by Van der Waal's forces, (steric) interdigitation of amphiphilic peptides, especially peptide helices, or peptides bearing opposite charges of amino acid residues. The correspondingly effective peptides are called above and below interactive or intercalating peptides.
The amphiphilic peptides consist of up to 50 amino acids. Preferrably they consist of 10 to 30 amino acids. In a preferred embodiment of the invention the interactive peptide is a peptide helix bundle (comprising of a helix, a turn and another helix, see above). In another embodiment the interactive peptide is a leucine zipper consisting of a peptide having several repeating amino acids, in which every seventh amino acid is a leucine residue. In other cases according to the invention the peptide bear positively or negatively charged residues, e.g. lysine (positively charged) or glutamic acid (negatively charged) in a way that this peptide can bind to another peptide (of a second monomeric unit) bearing opposite charges.
The Fv-fragment and the intercalating peptide are linked together either directly or by a linker peptide, preferrably by a linker peptide. In a preferred embodiment the linker peptide is a hinge region sequence of an antibody.
As defined, the Fv-fragment consists of the V.sub.L and V.sub.H region of an antibody. The Fv-fragment according to the invention is preferrably a single chain fragment. Single chain fragments can be obtained by standard techniques using standard linker molecules.
Furthermore, object of the invention is a dimeric fusion protein essentially consisting of two monomeric fusion proteins, wherein the linkage of the monomeric units bases on noncovalent interaction of identical or different peptides, characterized in that at least one monomeric unit is an antibody-Fv-fragment fusion protein as defined above.
If the dimer contains two Fv-fragments, the Fv-fragments may be the same (identical antigen binding sites) or may be different (different antigen binding sites). In these cases mono- and bispecific (Fv)-miniantibodies can be obtained. According to the invention bispecific mini-antibodies are preferred.
The interactive peptides may be the same or may be different; preferrably, they are identical. The intercalating peptides may be associated in parallel or in antiparallel fashion.
Object of the invention is, therefore, above all, a dimeric fusion protein consisting of two Fv-fragments with different specificity (antigen binding sites) and identical intercalating helix peptides, the antibody fragments and the peptides are linked together by a hinge region sequence.
Furthermore, object of the invention is a dimer consisting of a monomeric unit containing a Fv-fragment and another monomeric unit wherein the Fv-Fragment was replaced by a non-antibody peptide. The non-antibody peptide may be a toxin, like ricin, a chelator- or metal binding peptide, or an enzyme (e.g. marker enzyme), or a peptide bearing a detectable label (e.g. a radioisotope).
The non-antibody peptide can also bear a corresponding binding site for said groups, binding sites directed to T-cells or T-cell fragments included.
Furthermore, the invention relates to monomers and diners, as defined above, wherein the interactive peptide(s) is (are) additionally fused at the C-terminus to target proteins/peptides as mentioned above, the corresponding binding sites included. Thus, the resulting fusion proteins and miniantibodies, respectively, are multifunctional.
The invention relates, furthermore, to a process for preparation of a monomeric antibody fusion protein as defined above, characterized in that the genes coding for the Fv-fragment, the interactive peptide and, if desired, the linking peptide are cloned into one expression plasmid, a host cell is transformed with said expression plasmid and cultivated in a nutrient solution, and the monomeric fusion protein is expressed in the cell or secreted into the medium.
Object of the invention is, finally, a process for preparation of a dimeric fusion protein as defined above, characterized in that the genes coding for the complete monomeric fusion proteins or parts of it are cloned at least into one expression plasmid, a host cell is transformed with said expression plasmid(s) and cultivated in a nutrient solution, and either the complete dimeric fusion protein is expressed in the cell or into the medium, or the monomeric fusion proteins are separately expressed and the noncovalent linkage between the two monomeric units is performed in the medium or in vitro, and in the case that only parts of the fusion proteins were cloned, protein engineering steps are additionally performed according to standard techniques.
The dimeric Fv-fragments containing fusion proteins according to the invention show a high avidity against corresponding antigens and a satisfying stability. These novel bivalent or bifunctional molecules can be prepared as folded and assembled molecules in E. coli. These miniantibodies are compatible with functional expression by secretion.